Application of free flow electrophoresis to the preparative purification of basic proteins from an E. coli cell...

The application of free flow electrophoresis (FFE) to the purification of a basic protein from a complex protein mixture was investigated. For this purpose lysozyme (E.C.3.2.1.17) from hen egg white, serving as a model for a basic protein, was added to a crude E. coli cell extract and reisolated. For three techniques of FFE (zone electrophoresis, isoelectric focusing and field step electrophoresis), suitable electrolyte systems were developed. The purity, purification factor, recovery and throughput were determined for the optimized experiments. A combination of field step electrophoresis and zone electrophoresis gave the best purification factor (9.5) and the highest recovery (95%). The purification factors achieved in zone electrophoresis and isoelectric focusing were comparable to each other and ranged from 3.5 to 4.75. In isoelectric focusing, 94% of the enzyme activity was recovered. Zone electrophoresis gave recoveries of 82% and 87%, respectively. Purities of more than 95% were achieved with all the techniques described. With the exception of zone electrophoresis, all the techniques effected a concentration of the enzyme during the separation. Zone electrophoresis and field step electrophoresis were very simple in application.